Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item Development of a laboratory model to assess the removal of biofilm from interproximal spaces by powered tooth brushing(2002-11) Adams, Heather; Winston, Matthew T.; Heersink, Joanna; Buckingham-Meyer, Kelli; Costerton, J. William; Stoodley, PaulPurpose: To develop an interproximal laboratory model to compare the potential effectiveness of powered brushing to remove biofilm plaque from interproximal spaces beyond the reach of bristles. Materials and Methods: Streptococcus mutans biofilms were first grown onglass microscope slides in a drip-flow reactor. The slides were removed and positioned in the interproximal model. Each slide was exposed to 15 seconds powered brushing with either the Sonicare® Elite or the Braun Oral-B 3D Excel. The thickness of the biofilm was measured with confocal microscopy at various distances from the bristle tips. Results: The Sonicare® Elite reduced the thickness of biofilm by 57% at a distance of 0-5 mm from the bristle tips, 46% at 5-10 mm and 43% at 10-15 mm, relative to biofilm in areas unexposed to brushing. All reductions in thickness were statistically significant (P< 0.01). The Braun Oral-B 3D reduced the biofilm thickness by 16%, 13%, and 19% at the same distances respectively, but the thickness reductions were not statistically significant from those in the unexposed areas (P> 0.1).Item Monitoring of microbial souring in chemically treated, produced-water biofilm systems using molecular techniques(2005-04) Kjellerup, B. V.; Veeh, Richard Harold; Sumithrarathne, P.; Thomsen, T. R.; Buckingham-Meyer, Kelli; Frolund, B.; Sturman, Paul J.The identification of bacteria in oil production facilities has previously been based on culture techniques. However, cultivation of bacteria from these often-extreme environments can lead to errors in identifying the microbial community members. In this study, molecular techniques including fluorescence in situ hybridization, PCR, denaturing gradient gel electrophoresis, and sequencing were used to track changes in bacterial biofilm populations treated with nitrate, nitrite, or nitrate+molybdate as agents for the control of sulfide production. Results indicated that nitrite and nitrate+molybdate reduced sulfide production, while nitrate alone had no effect on sulfide generation. No long-term effect on sulfide production was observed. Initial sulfate-reducing bacterial numbers were not influenced by the chemical treatments, although a significant increase in sulfate-reducing bacteria was observed after termination of the treatments. Molecular analysis showed a diverse bacterial population, but no major shifts in the population due to treatment effects were observed.Item Spatial patterns of DNA replication, protein synthesis and oxygen concentration within bacterial biofilms reveal diverse physiological states(2007-03) Rani, Suriani A.; Pitts, Betsey; Beyenal, Haluk; Veluchamy, Raaja R. A.; Lewandowski, Zbigniew; Davison, William M.; Buckingham-Meyer, Kelli; Stewart, Philip S.It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 μm and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms.Item Comparative evaluation of biofilm disinfectant efficacy tests(2007-08) Buckingham-Meyer, Kelli; Goeres, Darla M.; Hamilton, Martin A.Regulatory agencies are receiving registration applications for unprecedented, antibiofilm label claims for disinfectants. Reliable, practical, and relevant laboratory biofilm test methods are required to support such claims. This investigation describes the influence of fluid dynamics on the relevancy of a laboratory test. Several disinfectant formulations were tested using three different biofilm testing systems run side-by-side: the CDC biofilm reactor system that created turbulent flow (Reynolds number between 800 and 1850), the drip flow biofilm reactor system that created slow laminar flow (Reynolds number between 12 and 20), and the static biofilm system that involved no fluid flow. Each comparative experiment also included a dried surface carrier test and a dried biofilm test. All five disinfectant tests used glass coupons and followed the same steps for treatment, neutralization, viable cell counting, and calculating the log reduction (LR). Three different disinfectants, chlorine, a quaternary ammonium compound, and a phenolic, were each applied at two concentrations. Experiments were conducted separately with Pseudomonas aeruginosa and Staphylococcus aureus and every experiment was independently repeated. The results showed that biofilm grown in the CDC reactor produced the smallest LR, the static biofilm produced the largest LR, and biofilm grown in the drip flow reactor produced an intermediate LR. The differences were large enough to be of practical importance. The dried surface test often produced a significantly higher LR than the tests against hydrated or dried biofilm. The dried biofilm test produced LR values similar to those for the corresponding hydrated biofilm test. These results show that the efficacy of a disinfectant must be measured by using a laboratory method where biofilm is grown under fluid flow conditions similar to the environment where the disinfectant will be applied.Item A method for growing a biofilm under low shear at the air–liquid interface using the drip flow biofilm reactor(2009-04) Goeres, Darla M.; Hamilton, Martin A.; Beck, Nicholas A.; Buckingham-Meyer, Kelli; Hilyard, Jackie D.; Loetterle, Linda R.; Lorenz, Lindsey A.; Walker, Diane K.; Stewart, Philip S.This protocol describes how to grow a Pseudomonas aeruginosa biofilm under low fluid shear close to the air–liquid interface using the drip flow reactor (DFR). The DFR can model environments such as food-processing conveyor belts, catheters, lungs with cystic fibrosis and the oral cavity. The biofilm is established by operating the reactor in batch mode for 6 h. A mature biofilm forms as the reactor operates for an additional 48 h with a continuous flow of nutrients. During continuous flow, the biofilm experiences a low shear as the media drips onto a surface set at a 101 angle. At the end of 54 h, biofilm accumulation is quantified by removing coupons from the reactor channels, rinsing the coupons to remove planktonic cells, scraping the biofilm from the coupon surface, disaggregating the clumps, then diluting and plating for viable cell enumeration. The entire procedure takes 13 h of active time that is distributed over 5 d.Item Checking the validity of the harvesting and disaggregating steps in laboratory tests of surface disinfectants(2009-11) Hamilton, Martin A.; Buckingham-Meyer, Kelli; Goeres, Darla M.A chemical disinfectant against surface-associated bacteria typically uses carriers (e.g., glass disks)that are purposely contaminated with bacteria prior to disinfection. After disinfection, the bacteria are harvested by mechanically separating them from the carrier surface to form a suspension of cells in a dilution tube. Bacterial clumps in the tube are disaggregated using mechanical or chemical techniques, thereby creating a well-mixed suspension of single cells suitable for enumeration. Efficacy is quantified by comparing the viable cell count for a disinfected carrier to the viable cell count for sham-disinfected (control) carrier. A test is said to be biased (invalid) if the observed efficacy measure is systematically higher or lower than the true efficacy. It is shown here for the first time that the bias attributable to the harvesting and disaggregating steps of a disinfectant test can be measured. For some conventional biofilm harvesting and disaggregating techniques, laboratory checks showed either negligible bias or important bias, depending on the disinfectant. Quantitative bias checks on the harvesting and disaggregating steps are prudent for each combination of carrier material, microorganism, and disinfectant. The quantitative results should be augmented by microscopic examination of harvested disinfected and control carriers and of the disaggregated suspensions.